1. Field of the Invention
This invention relates to cell membrane-associated DNA binding proteins (termed DNA-R herein) from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel mammalian DNA-R gene. The invention also relates to the construction of recombinant expression constructs comprising cDNA of this novel DNA-R gene, said recombinant expression constructs being capable of expressing DNA-R protein in cultures of transformed prokaryotic and eukaryotic cells. Production of the receptor protein in such cultures is also provided, as well as the production of fragments thereof having biological activity. The invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of the novel DNA-R protein. The invention also provides cultures of such cells producing this DNA-R protein for the characterization of novel and useful drugs. Antibodies against and epitopes of this novel DNA-R protein are also provided by the invention.
2. Background of the Invention
Extracellular DNA is a potent biological signal, being capable of initiating a wide range of immune responses in vivo and in vitro; including cytokine production, influx of neutrophils, IgM secretion, B-cell proliferation and enhanced natural killer activity. These properties of extracellular DNA enable naked DNA to be used as vaccines, in some instances. In addition, extracellular DNA has been used to introduce new genetic information into cells, both in vivo and in vitro.
One important aspect of extracellular DNA transfer into mammalian cells is gene therapy. Gene transfer therapy offers the potential for treatment of a variety of diseases. The ability to provide safe, efficient, and selective in vivo gene delivery will be a critical component of future protocols. Gene transfer by injection of either plasmid DNA or DNA/liposome complexes has been demonstrated to be safe and permits expression of gene products. The uptake of DNA/liposome complexes does not depend upon specific cell-surface receptors while the mechanism mediating uptake of plasmid DNA by cells remains unknown.
In order to realize the full potential of this technology, safe delivery and efficient transgene expression of DNA in selected tissues and cells must be achieved. One approach to target DNA to tissue is the use of a receptor-mediated mechanism for the binding and internalization of DNA. Viral (retrovirus, adenovirus, adeno-associated virus) delivery of DNA to cells is via a receptor-mediated mechanism, however this technique has limited in vivo clinical application. Viral vectors have been most frequently used for ex vivo gene therapy, but the technical problems associated with transplanting transduced cells remain a serious obstacle. In addition, viral vectors have the potential to lead to virus infection or to induce an immune response against antigenic viral coat proteins.
Non-viral methods of gene delivery include liposomes, the so-called “gene gun”, and direct injection. Gene transfer with liposomes has been shown to result in uptake and expression of DNA. Although DNA/liposomes are effectively taken up and the cDNA on the plasmid expressed, the process is believed to be nonspecific with limited possibility of targeting selected tissue. An alternative is to administer plasmid DNA directly, without a delivery system. Cells lines in tissue culture have demonstrated in vitro uptake of plasmid DNA and the expression of the transgene on the plasmid. It has also been shown that DNA, injected directly in vivo, has been taken up and the encoded genes have been expressed. While this approach has been shown to be a safe and free from problems associated with DNA delivery by viruses, the therapeutic potential of this technology is often limited by poor transgene expression from plasmid DNA in many tissues. In addition, the mechanism by which plasmid DNA is bound and internalized into cells is not well established. Knowledge of the mechanism of plasmid DNA binding to the cell surface, and how DNA is internalized and expressed, will be critical to enhancing transgene methods that also have the potential to target selected tissues.
Antisense oligonucleotides (ODN) are another form of extracellular DNA of great importance. ODN are considered potential therapeutic agents against various pathogens and oncogenes due to their ability to specifically inhibit gene expression. When injected into tissues, ODN are internalized by cells and bind to complementary region of mRNA to inhibit translation of proteins in a highly specific manner. Different antisense ODN to HIV RNA have been shown to inhibit the infectivity of the virus in cultured human leukemia cells. Although human clinical trials using ODN to treat AIDS and other diseases are ongoing, the lack of a precise understanding of where and how gene expression is effected hinders the optimization of this technique.
Extracellular DNA is also associated with human diseases, such as cystic fibrosis. Cystic fibrosis (CF) is the most common lethal genetic disease in North America. It affects one in 2500 live births and affected individuals have a median life expectancy of 28 years (Davis et al., 1996, Amer. J. Respir. Crit Care Med. 157: 1234–1239). There is a growing body of evidence showing that inflammation, particularly the injurious products of neutrophils, may be responsible for lung damage (Doring, 1997, Ped. Pulmonol. Supp. 16: 271–272); it is now recognized that most of the morbidity and over 90% of the mortality results from chronic progressive inflammation of the lungs. Corticosteroids have abroad anti-inflammatory effect, particularly on neutrophils. A multicenter trial showed beneficial effects of oral corticosteroids on lung function. However, adverse effects such as growth retardation, glucose abnormalities and cataracts prelude this treatment as a long-term option (Eigen et al., 1995, J. Ped. 126: 515–523). The nonsteroidal anti-inflammatory drug, ibuprofen, has also been studied (Konstan et al., 1995, N. Engl. J. Med. 332: 848–854). The drug is beneficial, but continued monitoring is needed to determine the safety of long-term, high dose therapy. Other therapies that treat the injurious products of neutrophils, for example, antiproteases and antioxidants, are currently under investigation (Konstan, 1998, Clin. Chest Med. 19: 505–513).
The vicious airway fluid characteristic of CF can obstruct airflow and provides a viable growth medium for pathogenic bacteria, and cell lysis of these bacteria can produce extracellular DNA that causes inflammation. Recombinant human Dnase (rhDNase) has been clinical use since 1994 (Kontsan, 1998, ibid.). The rhDNase, administered by inhalation, has been used to cleave the extracelular airway DNA and reduce the viscosity of the airway fluid. Treatment with rhDNase produces a small improvement in lung function (Cramer & Bosso, 1996, Ann. Pharmacol. 30: 656–661). However, when treatment is stopped, patients can deteriorate to a point below their previous baseline (Bush, 1998, Ped. Pulmonol. 25: 79–82). In addition, a recent report showed that despite improvements in lung function, there were no changes in airway inflammation (Henry et al., 1998, Ped. Pulmonol. 26: 97–100). Although the DNA is broken down by the Dnase, it is not entirely degraded, and hydrolized fragments are still potentially immunostimulatory and can contribute to inflammation. Thus rhDNase may be masking the process of on-going lung destruction.
There are also a variety of conventional treatments for CF including physiotherapy, nutritional support and drugs (Bilton & Mahadeva, 1997, J. Royal Soc. Med. 90: Suppl. 31,2–5). Because the events that trigger and sustain inflammation in patients with CF are not clearly understood, a variety of approaches have been developed to treat different components of the disease. Antibiotics, anti-inflammatories, and therapies to reduce the viscosity of the airway fluid are all approaches that are being used and investigated. Aggressive antibiotic therapy has helped the acute control of infection, but rarely if ever are the bacteria in the airways of patients with CF completely eradicated. These pathogenic bacteria chronically stimulate and exacerbate inflammation. Although some of the currently-available treatments can help to alleviate symptoms and slow the progression of disease, none of the current treatments can prevent ultimate respiratory failure.
One important clinical observation is that greatly increased amounts of extracellular DNA, of host and bacterial origin, are present in the airway of patients with cystic fibrosis. Recent investigation has demonstrated that extracellular DNA, purified from sputum of patients with CF, will directly induce inflammation in the mouse lung (Schwartz et al., 1997, J. Clin. Invest. 100: 68–73). The DNA purified from the sputum of patients with cystic fibrosis has been shown to be composed primarily of host-derived DNA and only a small fraction appears to be bacterial DNA (Schwartz et al., 1997, ibid.). One possible explanation is that extracellular DNA binds to immune lung cells in the lungs and induces the secretion of pro-inflammatory cytokines and neutrophic migration to the lung, leading to severe airway inflammation. Extracellular DNA binding to immune cells in the lung, such as alveolar macrophages are stimulated to produce pro-inflammatory cytokines that recruit and activate neutrophils leading to inflammation. When these neutrophils undergo apoptosis and release their DNA the cycle is repeated and inflammation is maintained or increased. Thus, methods and reagents that block DNA binding to cytokine producing cells may therefore provide better treatment of CF patients than are currently available.
Although there have been several reports in the art that DNA could bind to cell surfaces (Bennett, 1993, Antisense Res. Develop. 3: 235–241; Bennett et al., 1986, J. Rheumatol. 13: 679–685; Gabor & Bennett, 1984, Biochem Biophys. Res. Commun. 122:1034–1039; Hefeneider et al., 1990, J. Invest. Dermatol. 94: 79S–84S; Bennett et al., 1987, J. Exp. Med. 166: 850–863; Bennett et al., 1991, Clin. Exp. Immunol. 86:374–379; Bennett et al., 1992, Clin. Exp. Immunol 90: 428–433; Bennett et al., 1985, J. Clin. Invest. 76: 2182–2190; Hefeneider et al., 1992, Lupus 1: 167–173; Hefeneider et al., 1992, Clin. Immunol. Immunopath. 63: 245–251; Reid & Chalson, 1979, Intl. Rev. Cytol. 60: 27–52; Lerner et al., 1971, Proc. Natl. Acad. Sci. USA 68: 1212–1216; Pancer et al., 1981, J. Immunol. 127: 98–104; Meinke & Goldstein, 1974, J. Molec. Biol. 86: 757–773; Sudar et al., 1986, Cell. Molec. Biol. 32: 87–91; Gasparro et al., 1990, Photochem & Photobiol. 52: 315–321; Emlen et al., 1988, Amer. J. Pathol. 133: 54–60), the art lacks an understanding of how cells mediate extracellular DNA binding. Thus, an understanding of the mechanisms by which eukaryotic cells, particularly mammalian cells, take up extracellular DNA would be important in improving a variety of biological processes.